Project Abstract Insufficient supply of donor pancreatic islets to meet the demand for transplants severely limits therapeutic options for Type 1 diabetic (T1D) patients in whom insulin therapy is insufficient to control their disease. And while there has been much recent success with the directed differentiation of human embryonic stem cells (hu-ESCs) and human induced pluripotent stem cells (hu-iPSCs) into pancreatic beta cells, the process is still quite imperfect and inefficient. To this end, we have shown that Conditional Reprogrammed (CR) cell culture technology can support the significant expansion of human islet-derived ?-cells to at least one million-fold amount. Just as importantly, our preliminary data indicates that CR-propagated human ?-cells can readily develop mature differentiation markers and functional phenotype in vitro. To develop this promising technology into a practical option for human ?-cell replacement therapy, we seek to enhance the proliferative capacity of CR-grown human ?-cells through the use of a focused screening approach that we have used with success with other CR-responsive cell types. The optimization of the post-CR expansion differentiation step is also a component of our proposal, using reproduced findings from other labs on how cell surface interactions can help drive human ?-cell maturation and functionality. The ultimate test of using CR technology to expand human ?-cells will be to use these cells in an animal model of T1D and to assess their ability to modulate the disease pathology; a return to euglycemia and production of human C- peptide in the transplant-receiving mice will be the positive hallmark of success. Successful completion of these specific research aims will position us to propose a Phase II SBIR application focused on the preclinical development of CR technology into GLP efficacy and safety testing and cGMP-compliant cell manufacturing.